Agarose gel electrophoresis is an easy way to separate
DNA fragments by their sizes and visualize them. It is a common diagnostic
procedure used in molecular biological labs.

Electrophoresis:

The technique of electrophoresis is based on the fact that
DNA is negatively charged at neutral pH due to its phosphate backbone. For
this reason, when an electrical potential is placed on the DNA it will move
toward the positive pole:

Figure 1

The rate at which the DNA will move toward the positive
pole is slowed by making the DNA move through an agarose
gel. This is a buffer solution (which maintains the proper pH and salt concentration)
with 0.75% to 2.0% agarose added. The agarose forms a porous lattice in
the buffer solution and the DNA must slip through the holes in the lattice in order to move toward the positive
pole. This slows the molecule down. Larger molecules will be slowed down
more than smaller molecules, since the smaller molecules can fit through
the holes easier. As a result, a mixture of large and small fragments of
DNA that has been run through an agarose gel will be separated by size.
This is a graphic representation of an agarose gel made by "running"
DNA molecular weight markers, an isolated plasmid, and the same plasmid
after linearization with a restriction enzyme:

These gels are visualized on a U.V. trans-illuminator by
staining the DNA with a fluorescent dye (ethidium bromide). The DNA molecular
weight marker is a set of DNA fragments of known molecular sizes that are
used as a standard to determine the sizes of your unknown fragments.

If you click on the figure you will see a short movie that
simulates the movement of the DNA bands through the gel. When looking at
the video, note that bands of a low molecular weight move very quickly through
the gel while high molecular weight bands move very slowly.

Figure 2

Interpretation:

Much information can be derived from this gel. As you read
the text below, refer back to figure 2.

1.) By looking at the migration of the DNA molecular weight
standards, you can tell that the migration of DNA through an agarose gel
is not linear with respect to size. If you graphed the distance traveled
vs. the molecular weight of the fragment, you would see that there is a
logarithmic relationship (i.e. small fragments travel much faster than large
fragments).

2.) You can see that there is a big difference between
the way a plasmid as isolated from the alkaline lysis prep will run vs.
this same plasmid after it is cut with a restriction enzyme and linearized.
This is because the plasmid will be found in many different supercoiled
forms in the bacteria. When you isolate plasmid from a bacterial culture,
you isolate all the different supercoiled forms of the plasmid, and each
will migrate differently on the gel, giving you three major bands and many
minor bands. When this mixture of supercoiled plasmids is cut with a restriction
enzyme, the different forms linearize and unwind. As a result they all become
identical and run at the same rate, and you see only one band on the gel.

3.) The molecular size of an unknown piece of DNA can be
estimated by comparison of the distance that it travels with that of the
molecular weight standards. This is only true for linear DNA. None of the
supercoiled forms will migrate at a rate relative to linear DNA, which means
that you can't use the DNA markers to estimate the molecular weight of a
circular DNA molecule. To estimate the molecular weight of a plasmid, you
must first linearize it. By looking at the gel above, the molecular size
of the plasmid can be estimated at approximately 3.0 kilobases
(kb). A more accurate estimate can be found by graphing the molecular weight
of the standards (in base pairs) vs. the distance traveled on semi-log paper
and using this graph to determine the molecular weight of the unknown. You
will do this at the end of this experiment. Molecular size is the most important
information derived from the agarose gel and the usual reason for running
a gel.

In this experiment, you will linearize the plasmid that
you isolated last week with a restriction enzyme. Then you will run this
linearized plasmid on an agarose gel with the uncut version and a DNA marker
to determine the size of your plasmid + insert, which will give you an estimate
of the size of your insert.

Procedure:

1.) Put together the following reaction mixture for the
restriction digestion:

14.5 ul water

2.0 ul 10X Rest. Enzyme buffer

3.0 ul plasmid DNA solution (from last week)

0.5 ul Restriction Enzyme (eg., HindIII)

20.0 ul Total

Add the enzyme last, and always keep it on ice. The enzyme
you will use will depend on the plasmid that you have, and will be told
to you during class. 0.5 ul can't be measured with your pipetman. You must
estimate it by the way it will look in the pipet tip (instruction will be
given in class). Be sure to use a clean tip when taking the enzyme out of
the tube. Put this reaction at 37oC for 45 minutes.

2.) When the digestion is complete, prepare to load the
gel. In a new tube, place 17.0 ul of H2O and 3.0 ul of uncut
plasmid DNA. Add 2.0 ul dye to each of the three sample tubes (DNA markers,
uncut plasmid, and digested plasmid). Load 20.0 ul of DNA marker in to one
well of the gel. Do this by sucking the solution into the pipet tip, placing
the tip in the top of the well, and gently expelling the liquid into the
well. The dye buffer in the DNA marker and samples contains glycerol which
makes it more dense than H2O. This will cause the liquid to sink
to the bottom of the well. Load 20.0 ul of the uncut plasmid and the restriction
digestion.

3.) Turn on the power supply and electrophorese the samples
at 110 V (Warning- be careful of the high voltage or you will be
set down on your butt dramatically.) Electrophorese the samples until the
dark blue dye is about 2 cm from the bottom of the gel

4.) Stain the gel by incubating it for 8 min in an ethidium
bromide solution.

WARNING!

Ethidium bromide is very carcinogenic. Handle
this gel only while wearing gloves. Never put unprotected fingers in the
gel buffer solution.

5.) I will move the gel onto the U.V. trans illuminator
and take a picture of it.

6.) I will denature the ethidium bromide by placing the
gel in potassium permanganate solution for 5 minutes, then discard it.

Calculations:

On a piece of semi-log graph paper, plot the log of the
molecular weight each standard vs. distance traveled from the well (based
on measurements made from the picture of the gel). The sizes of the molecular
weight standards may be different than in figure 2 and will be given to
you during lab. Draw a line connecting the points (although the line won't
be linear). From this you should be able to determine the molecular size
of the linearized plasmid. Note the difference between the way the linearized
DNA ran and the way the uncut plasmid appears. If you have multiple bands
in the digested plasmid lane, see if some of them are of the same size as
bands in the uncut lane; you may have incomplete digestion of your plasmid
due to a sub-optimal purification (i.e., a dirty plasmid prep). Since each
of the vector plasmids (without the insert) is 3.0 kb, you can determine
the size of the insert in your plasmid by subtracting 3.0 from the size
estimated from the gel.